Neuroblastoma, one of the most common forms of childhood cancer has been treated with limited success with various antimetabolites used singly, sequentially or in combination. This tumor has been cloned from the mouse and adpated to tissue culture. During the past two years a series of new folic acid analogues and other inhibitors of pyrimidine and purine synthesis have been studied in our laboratory. This experimental system has allowed the exquisite quantitation of sensitivites to drug combinations in a sensitive parental clone of this cell line and in various drug resistant mutants which have isolated. The molecular mechanisms by which mutants effect resistance to these drugs have been characterized by correlating the degree of resistance to growth inhibition with changes in the mutants transport of these drugs or in the in vitro activities of enzymes affecred by them, emphasizing thymidylate synthetase and dihydrofolate reductase. Our investigation has led us to a newly purified antifolate which uniquely inhibits both thymidylate synthetase and dihydrofolate reductase. We propose to evaluate this new drug, to examine in greater depth the mechanisms of antimetabolite resistance in neuroblastoma which we have now outlined, to determine the homogeneity of these mechanisms, and to test further compounds which might act synergestically with those already examined. These studies are intended to provide a better understanding of the patterns of drug resistance, allowing more effective chemotherapy regimens to be formulated.